Single-strand conformational polymorphism analysis of the M(r) 170,000 isozyme of DNA topoisomerase II in human tumor cells

Five cell lines selected for resistance to the cytotoxicity of inhibitors of DNA topoisomerase II have point mutations in the gene that codes for the M(r) 170,000 form of this enzyme. In each case, the mutation results in an amino acid change in or near an ATP binding sequence of the M(r) 170,000 isozyme of topoisomerase II. We used single-strand conformational polymorphism analysis to screen for similar mutations in other drug-resistant cell lines or in leukemic cells from patients previously treated with etoposide or teniposide. We also analyzed the region of the gene that codes for amino acids adjacent to the tyrosine at position 804 of topoisomerase II which binds covalently to DNA. CEM/VM-1, CEM/VM-1-5, and HL-60/AMSA human leukemic cell lines were used as controls; 3 of 3 known mutations were detected by migration differences of polymerase chain reaction products from the RNA extracted from these three lines. A previously unknown mutation was found in the tyrosine 804 region of the M(r) 170,000 topoisomerase II expressed by CEM/VM-1 and CEM/VM-1-5 cells. Sequence analysis showed that substitution of a T for a C at nucleotide 2404 resulted in an amino acid change of a serine for a proline at amino acid 802. No mutations in any of the ATP binding sequences or in the tyrosine 804 region were detected in polymerase chain reaction products from RNA extracted from human leukemia HL-60/MX2 or CEM/MX1 cells (both cell lines selected for resistance to mitoxantrone) or in human myeloma 8226/Dox1V cells (selected for resistance by simultaneous exposure to doxorubicin and verapamil). No mutations were detected in polymerase chain reaction products from RNA extracted from blasts of 15 patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide. We conclude that: (a) single-strand conformational polymorphism analysis is useful for screening for mutations in topoisomerase II; (b) resistance to the cytotoxicity of inhibitors of DNA topoisomerase II is not always associated with mutations in ATP binding sequences or the active site tyrosine region of M(r) 170,000 topoisomerase II; and (c) mutations similar to those detected in drug resistant cells selected in culture have not been identified in blast cells from patients with relapsed acute lymphocytic leukemia, previously treated with etoposide or teniposide.     

Drug-specific sites of topoisomerase II DNA cleavage in Drosophila chromatin: heterogeneous localization and reversibility

DNA cleavage stimulated by different topoisomerase II inhibitors shows in vitro a characteristic sequence specificity. Since chromatin structure and genome organization are expected to influence drug-enzyme interactions and repair of drug-induced DNA lesions, we investigated topoisomerase II DNA cleavage sites stimulated by teniposide (VM-26), 4-demethoxy-3'-deamino-3'-hydroxy-4'-epi-doxorubicin (dh-EPI, a doxorubicin derivative), 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and amonafide in the histone gene locus and satellite III DNA of Drosophila cells with Southern blottings and genomic sequencing by primer extension. VM-26 stimulated cleavage in the satellite III DNA, whereas the other studied drugs did not. All four drugs stimulated cleavage in the histone gene cluster, but they yielded drug-specific cleavage intensity patterns. Cleavage sites by dh-EPI and VM-26 were sequenced in the histone H2A gene promoter and were shown to be distinct. DNA cleavage analysis in cloned DNA fragments with Drosophila topoisomerase II showed that drugs stimulated the same sites in vivo and in vitro. Strand cuts were in vivo staggered by 4 bases, and base sequences at major dh-EPI and VM-26 sites completely agreed with known in vitro drug sequence specificities. Moreover, DNA cleavage reverted faster in the satellite III than in the histone repeats. While stimulating similar levels of DNA breakage in bulk genomic DNA, dh-EPI and VM-26 markedly differed for cleavage extent and reversibility in specific chromatin loci. The results demonstrate a high heterogeneity in the localization, extent, and reversibility of drug-stimulated DNA cleavage in the chromatin of living cells.     

Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells

The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes. GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S. De Jong et al., Cancer Res., 50: 304-309, 1990). In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed. Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug. Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells. Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells. On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA. Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance. Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4. The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting. No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized. These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.     

A carbamate analogue of amsacrine with activity against non-cycling cells stimulates topoisomerase II cleavage at DNA sites distinct from those of amsacrine

AMCA (methyl N-[4-(9-acridinylamino)-2-methoxyphenyl]carbamate hydrochloride), an amsacrine analogue containing a methylcarbamate rather than a methylsulphonamide side chain, contrasts with amsacrine, doxorubicin and etoposide in its relatively high cytotoxicity against non-cycling tumour cells. AMCA bound DNA more tightly than amsacrine, but the DNA base selectivity of binding, as measured by ethidium displacement from poly[dA-dT].[dA-dT] and poly[dG-dC].[dG-dC], was unchanged. AMCA-induced topoisomerase cleavage sites on pBR322, C-MYC and SV40 DNA were investigated using agarose or sequencing gels. DNA fragments were end-labelled, incubated with purified topoisomerase II from different mammalian sources and analysed after treatment with sodium dodecylsulphate/proteinase K. AMCA stimulated the cleavage activity of topoisomerase II, but the DNA sequence selectivity of cleavage was different from that of amsacrine and other topoisomerase inhibitors. It was similar to that of the methoxy derivative of AMCA, indicating that the changed specificity resulted from the carbamate group rather than from the methoxy group. The pattern of DNA cleavage induced by AMCA was similar for topoisomerase II alpha and II beta.     

Amsacrine-promoted DNA cleavage site determinants for the two human DNA topoisomerase II isoforms alpha and beta

Site-specific DNA cleavage by topoisomerase II (EC 5.99.1.3) is induced by many antitumour drugs. Although human cells express two genetically distinct topoisomerase II isoforms, thus far the role and determinants of drug-induced DNA cleavage have been examined only for alpha. Here we report the first high-resolution study of amsacrine (mAMSA) induced DNA breakage by human topoisomerase II beta (overexpressed and purified from yeast) and a direct comparison with the recombinant alpha isoform. DNA cleavage in plasmid pBR322 and SV40 DNA was induced by alpha or beta in the absence or presence of the antitumour agent mAMSA, and sites were mapped using sequencing gel methodology. Low-resolution studies indicated that recombinant human alpha promoted DNA breakage at sites akin to those of beta, although some sites were only cleaved by one enzyme and different intensities were observed at some sites. However, statistical analysis of 70 drug-induced sites for beta and 70 sites for alpha revealed that both isoforms share the same base preferences at 13 positions relative to the enzyme cleavage site, including a very strong preference for A at +1. The result for recombinant alpha isoform is in agreement with previous studies using alpha purified from human cell lines. Thus, alpha and beta proteins apparently form similar ternary complexes with mAMSA and DNA. Previous studies have emphasized the importance of DNA topoisomerase II alpha; the results presented here demonstrate that beta is an in vitro target with similar site determinants, strongly suggesting that beta should also be considered a target of mAMSA in vivo.     

Genomic fluidity is a necessary event preceding the acquisition of tumorigenicity during spontaneous neoplastic transformation of WB-F344 rat liver epithelial cells

We have shown that both DNA topoisomerase (topo) IIalpha and beta are in vivo targets for etoposide using a new assay which directly measures topo IIalpha and beta cleavable complexes in individual cells after treatment with topo II targeting drugs. CCRF-CEM human leukemic cells were exposed to etoposide for 2 hr, then embedded in agarose on microscope slides before cell lysis. DNA from each cell remained trapped in the agarose and covalently bound topo II molecules from drug-stabilized cleavable complexes remained associated with the DNA. The covalently bound topo II was detected in situ by immunofluorescence. Isoform-specific covalent complexes were detected with antisera specific for either the alpha or beta isoform of topo II followed by a fluorescein isothiocyanate-conjugated second antibody. DNA was detected using the fluorescent stain Hoechst 33258. A cooled slow scan charged coupled device camera was used to capture images. A dose-dependent increase in green immunofluorescence was observed when using antisera to either the alpha or beta isoforms of topo II, indicating that both isoforms are targets for etoposide. We have called this the TARDIS method, for trapped in agarose DNA immunostaining. Two key advantages of the TARDIS method are that it is isoform-specific and that it requires small numbers of cells, making it suitable for analysis of samples from patients being treated with topo II-targeting drugs. The isoform specificity will enable us to extend our understanding of the mechanism of interaction between topo II-targeting agents and their target, the two human isoforms.     

Evaluation of bleached kraft mill process water using Microtox(R), Ceriodaphnia dubia, and Menidia beryllina toxicity tests

We show herein that human DNA topoisomerase II beta is functional in yeast. It can complement a yeast temperature-sensitive mutation in topoisomerase II. The effect on human topoisomerase II beta of a number of topoisomerase II inhibitors was analysed in a yeast in vivo system and compared with that of human topoisomerase II alpha and wild-type yeast topoisomerase II. A drug permeable yeast strain (JN394 top2-4) was used to analyse the in vivo effects of known anti-topoisomerase II agents on human topoisomerase II beta transformants. A parallel analysis on human topoisomerase II alpha transformants provides the first in vivo analysis of the responses of yeast bearing the individual isoforms to these drugs. The strain was analysed at 35 degrees C, a non-permissive temperature at which only plasmid-borne topoisomerase II is active. A shuttle vector with either human topoisomerase II beta, human topoisomerase II alpha or yeast topoisomerase II under the control of a GAL1 promoter was used. The key findings were that amsacrine produced comparable levels of cell killing with both alpha and beta, whilst etoposide, doxorubicin and mitoxantrone produced higher degrees of cell killing with alpha than with beta or yeast topoisomerase II. Merbarone had the greatest effect on the yeast strain bearing plasmid-borne yeast topoisomerase II. Suramin, quercetin and genistein showed little cell killing in this system. This yeast in vivo system provides a powerful way to analyse the effects of anti-topoisomerase II agents on transformants bearing the individual human isoforms. This system also provides a means of analysing putative drug-resistance mutations in human topoisomerase II beta or to select for drug-resistance mutations in human topoisomerase II beta.     

Characterization of DNA topoisomerase II alpha/beta heterodimers in HeLa cells

In mammalian cells, DNA topoisomerase II is the product of two distinct genes encoding the alpha and beta isoforms of the enzyme. Besides homodimeric topoisomerase IIalpha and IIbeta, we have recently shown that alpha/beta heterodimers constitute a third population of topoisomerase II in HeLa cells. We found that topoisomerase II heterodimers are not restricted to HeLa cells but exist in different mammalian cell types, and up to 25% of the total topoisomerase IIbeta population is involved in heterodimer formation. Studies of topoisomerase II phosphorylation in HeLa cells show that heterodimers are phosphorylated in vivo to a significantly lower level compared to homodimeric alpha enzymes, but in contrast to the latter neither heterodimers nor topoisomerase IIbeta homodimers coprecipitate together with a kinase activity that is able to mediate their phosphorylation. However, both enzymes can still be phosphorylated by exogenously added casein kinase II. The differential phosphorylation of topoisomerase II heterodimers suggests an alternative regulation of this topoisomerase II subclass compared to the homodimeric topoisomerase IIalpha counterparts.     

The responses of secondary endings of cat soleus muscle spindles to succinyl choline

In this report we examine biochemical and genetic alterations in DNA topoisomerase II (topoisomerase II) in K562 cells selected for resistance in the presence of etoposide (VP-16). Previously, we have demonstrated that the 30-fold VP-16-resistant K/VP.5 cell line exhibits decreased stability of drug-induced topoisomerase II/DNA covalent complexes, requires greater ATP concentrations to stimulate VP-16-induced topoisomerase II/DNA complex formation, and contains reduced mRNA and protein levels of the M(r) 170,000 isoform of topoisomerase II, compared with parental K562 cells. K/VP.5 cells grown in the absence of VP-16 for 2 years maintained resistance to VP-16, decreased levels of topoisomerase II, and attenuated ATP stimulation of VP-16-induced topoisomerase II/DNA binding, compared with K562 cells. Sequencing of cDNA coding for two consensus ATP binding sites and the active site tyrosine in the K/VP.5 topoisomerase II gene indicated that no mutations were present in these domains. In addition, single-strand conformational polymorphism analysis of restriction fragments encompassing the entire topoisomerase II cDNA revealed no evidence of mutations in the gene for this enzyme in K/VP.5 cells. Nuclear extracts from K562 (but not K/VP.5) cells contained a heat-labile factor that potentiated VP-16-induced topoisomerase II/DNA covalent complex formation in isolated nuclei from K/VP.5 cells. Immunoprecipitated topoisomerase II from K/VP.5 cells was 2.5-fold less phosphorylated, compared with enzyme from K562 cells. Collectively, our data suggest that acquired VP-16 resistance is mediated, at least in part, by altered levels or activity of a kinase that regulates topoisomerase II phosphorylation and hence drug-induced topoisomerase II/DNA covalent complex formation and stability.     

Effects of DNA methylation on topoisomerase I and II cleavage activities

DNA methylation is deregulated during oncogenesis. Since several major anti-cancer drugs act on topoisomerases, we investigated the effects of cytosine methylation on topoisomerase cleavage activities. Both topoisomerase I and II cleavage patterns were modified by CpG methylation in c-myc gene DNA fragments. Topoisomerase II changes, mainly cleavage reduction, occurred for methylation sites within 7 base pairs from the topoisomerase II breaks and were different for VM-26 and azatoxin. For topoisomerase I, cleavage enhancement as well as suppression were observed. Using synthetic methylated oligonucleotides, we show that hemimethylation is sufficient to alter topoisomerase I activity. Cytosine methylation on the scissile strand within the topoisomerase I consensus sequence had strong effects. Cleavage was stimulated by methylation at position -4 and was strongly inhibited by methylation at position -3 (with position -1 being the enzyme-linked nucleotide). This inhibitory effect was attributed to the presence of a methyl group in the major groove, since the transition uracil to thymine also inhibited cleavage. Altogether these results suggest an interaction of topoisomerase I with the DNA major grove at positions -3 and -4. In addition, DNA methylation may have profound effects on the activity of topoisomerases and may alter the distribution of cleavage sites produced by anticancer drugs in chromatin.     

Cloning of cDNA encoding a novel mouse DNA topoisomerase III (Topo IIIbeta) possessing negatively supercoiled DNA relaxing activity, whose message is highly expressed in the testis

We cloned cDNA encoding a novel mouse homologue of DNA topoisomerase III (mTOP3beta). The nucleotide sequence contains an open reading frame of 863 amino acids, and the deduced molecular mass of the coded protein is 96.9 kDa. The overall sequence of mTOP3beta has a 48 and 36% identity with mouse TOP3alpha at the nucleotide and amino acid level, respectively. DNA topoisomerase IIIbeta was expressed using a baculovirus expression system and purified. The purified DNA topoisomerase IIIbeta had activity to relax negatively supercoiled DNA. Relaxation of supercoiled DNA was partial at 37 degreesC and complete relaxation was observed at higher temperatures. mTOP3beta mRNA was strongly expressed in the testis and relatively strongly in the brain. The levels of TOP3beta mRNA in the testis increased slightly 14 days and considerably 17 days after birth, when the cells in the pachytene phase begin to appear and increase.     

Physiological measures of right nostril breathing

DNA topoisomerases, nuclear enzymes that regulate DNA topology, are recognized as the primary targets of effective anti-tumor drugs. These enzymes may also have a role in the repair of DNA damage induced by alkylating agents and platinum compounds; therefore, their expression may be a determinant of tumor response to chemotherapy. Our study was undertaken in an attempt to establish a correlation between the enzyme expression and response of ovarian cancer to cisplatin-based chemotherapy. The expression of topoisomerase I, II alpha and II beta genes was assessed by RNase protection assay in tumor specimens obtained from 37 untreated patients with advanced epithelial ovarian cancer at initial surgery and from 13 pre-treated patients at subsequent laparotomy. The expression levels were compared with those found in 5 specimens from benign ovarian tissue and 5 specimens from normal ovarian tissue. The expression levels in untreated patients were used to establish a correlation with response to high-dose cisplatin therapy. A significant intertumor variability of mRNA expression was noted for all the genes examined. However, a comparison of median values indicated a remarkable increase of expression in malignant tumors over benign or normal tissues only for topoisomerase II alpha. This change is not related to alterations or amplification of topoisomerase II alpha gene. Interestingly, a correlation was found between tumor response to chemotherapy and the expression level of the isoform alpha (but not of topoisomerase II beta and topoisomerase I). The observed correlation suggests a contribution of the enzyme in determining tumor sensitivity. Alternatively, increased expression levels of the alpha isoenzyme gene in responsive tumors might reflect higher fractions of proliferating tumor cells that may be more drug-sensitive than resting cells.     

Relationship between amine-carboxyboranes and TNF alpha for the regulation of cell growth in different tumor cell lines

The amine-carboxyboranes were shown to be synergistic with tumor necrosis factor alpha (TNF alpha) in cytotoxicity and inhibition of DNA synthesis in select types of cancer cells depending on the presence of a TNF alpha high affinity receptor on the membrane of the cell. Initially both TNF alpha and the amine-carboxyboranes reduce the influx of calcium but later cause a significant increase intracellularly. This influx is not linked with the amine-carboxyborane activating the calcitonin receptor in the tumor cells. Neither the agents nor TNF alpha directly inhibits DNA topoisomerase II activity but both did cause decreased phosphorylation of the enzyme by protein kinase C (PKC). The two agents caused synergistic inhibition. This event correlated with increased DNA protein linked breaks, DNA fragmentation and cell death. These protein linked breaks are additive with etoposide's effects but the latter agent's mechanism is different than phosphorylation of topoisomerase II. There was no evidence that the DNA fragmentation was caused by a calcium induced endonuclease enzyme in these cancer cells. The low-molecular weight amine-carboxyboranes appear to play an identical function as TNF alpha in its role to cause DNA breaks and fragmentation to cause apoptosis.     

Further purification and characterization of a multienzyme complex for DNA synthesis in human cells

The 21 S complex of enzymes for DNA synthesis in the combined low salt nuclear extract-post microsomal supernatant from HeLa cells [Malkas et al. (1990) Biochemistry 29:6362-6374] was purified by poly (ethylene glycol) precipitation, Q-Sepharose chromatography, Mono Q Fast Protein Liquid Chromatography (FPLC), and velocity gradient centrifugation. The procedure gives purified enzyme complex at a yield of 45%. The 21 S enzyme complex remains intact and functional in the replication of simian virus 40 DNA throughout the purification. Sedimentation analysis showed that the 21 S enzyme complex exists in the crude HeLa cell extract and that simian virus 40 in vitro DNA replication activity in the cell extract resides exclusively with the 21 S complex. The results of enzyme and immunological analysis indicate that DNA polymerase alpha-primase, a 3',5' exonuclease, DNA ligase I, RNase H, and topoisomerase I are associated with the purified enzyme complex. Denaturing polyacrylamide gel electrophoresis of the purified enzyme complex showed the presence of about 30 polypeptides in the size range of 300 to 15 kDa. Immunofluorescent imaging analysis, with antibodies to DNA polymerase alpha,beta and DNA ligase I, showed that polymerase alpha and DNA ligase I are localized to granular-like foci within the nucleus during S-phase. In contrast, DNA polymerase beta, which is not associated with the 21 S complex, is diffusely distributed throughout the nucleoplasm.     

DNA topoisomerase II alpha expression is associated with alkylating agent resistance

Increased expression of DNA topoisomerase II alpha has been associated with resistance to certain DNA-damaging alkylating agents, but no causal relationship or mechanism has been established. To investigate this observation, we developed a model of topoisomerase II overexpression by transfecting a full-length Chinese hamster ovary topoisomerase II alpha into EMT6 mouse mammary carcinoma. Topoisomerase II alpha-transfected cell lines demonstrated continued topoisomerase II alpha mRNA and protein expression, which were undetectable in vector-only lines, in stationary phase (G0-G1). The topoisomerase II transfectants were approximately 5-10-fold resistant to the alkylating agents cisplatin and mechlorethamine. Upon release from G0-G1, the topoisomerase II transfectants demonstrated more rapid thymidine incorporation and shorter cell-doubling times than control cells. Purified topoisomerase II and nuclear extracts with topoisomerase II-decatenating activity bound to cisplatin-treated DNA with significantly greater affinity than to untreated DNA in a cisplatin concentration-dependent manner. These observations suggest that expression of topoisomerase II alpha may have a role in cellular resistance to antineoplastic alkylating agents. The mechanism for this may involve increased binding of topoisomerase II alpha to alkylating agent-damaged DNA.     

DNA gyrase and topoisomerase IV: biochemical activities, physiological roles during chromosome replication, and drug sensitivities

DNA gyrase and topoisomerase IV are the two type II topoisomerases present in bacteria. Though clearly related, based on amino acid sequence similarity, they each play crucial, but distinct, roles in the cell. Gyrase is involved primarily in supporting nascent chain elongation during replication of the chromosome, whereas topoisomerase IV separates the topologically linked daughter chromosomes during the terminal stage of DNA replication. These different roles can be attributed to differences in the biochemical properties of the two enzymes. The biochemical activities, physiological roles, and drug sensitivities of the enzymes are reviewed.     

Drastic reduction of topoisomerase II alpha associated with major acquired resistance to topoisomerase II active agents but minor perturbations of cell growth

V511 and V513 cell lines, derived from Chinese hamster V79 cells following alkylating agent mutagenesis and subsequent selection with VP-16, showed resistance to cytotoxicity and DNA strand breaks induced by topoisomerase (topo) II inhibitors and were resistant to VP-16-induced sister chromatid exchanges. They showed no amplification of the multidrug-resistant p-glycoprotein. In a kinetoplast-DNA decatenation assay, V511 and V513 showed 51% and 49% topo II activity relative to parental V79 cells, respectively. By western-blot analysis all three logarithmically growing cell lines showed similar levels of topo II beta (M(r) 180,000), which increased as cells progressed to quiescence. In contrast, immunoreactive levels of topo II alpha (M(r) 170,000) were 6.8% in V511 and 62.4% in V513 relative to V79. V511 showed drastically decreased topo II alpha in both log growth and quiescence. In a second approach, immunoreactive topo II was analyzed in different phases of the cell cycle in logarithmically growing cells fractionated by fluorescence-activated cell sorting. All cell lines demonstrated relatively stable topo II beta throughout the cell cycle. Topo II alpha showed little cell cycle variation in V79 or V513. However, in V511, it was only detectable at low levels in G2/M phase. When cell growth parameters were measured, V511 and V513 showed a 17% increase in cell doubling time relative to V79. These studies indicate that cells with a drastic reduction in topo II alpha (V511) or mutant topo II alpha (V513) but with normal levels of topo II beta show only minor perturbations of cell growth.